Search

Human endogenous retroviruses (HERVs) are genetic elements, derived from their exogenous retroviral counterpart by a process of germline infection and proliferation within the human genome, and their integration as proviruses led to the fixation and the vertical transmission, following Mendelian laws. HERVs currently make up ~8% of the genetic material, and some of them have been cooped for physiological functions. Otherwise, their activation in response to environmental factors has been associated with human pathological conditions. In the setting of neurodevelopmental disorders, HERVs have been proposed as contributing factors involved in Autism Spectrum Disorders (ASD), spanning the bridge between genetic susceptibility, environmental risk factors and immune response. We described a distinct expression profile of some HERV families and cytokines in lymphocytes from autistic children and in their mothers suggesting a close mother-child association in ASD. Moreover, in vitro treatment with an antiretroviral drug was able to restore the expression level of HERVs and cytokines providing new insights into the potential role of HERVs as biomarkers of ASD and raising the possibility of using HERVs expression as a therapeutic target for a tailored approach to patient care.

Maternal infections during pregnancy and the consequent maternal immune activation (MIA) are the major risk factors for autism spectrum disorder (ASD). Epidemiological evidence is corroborated by the preclinical models in which MIA leads to ASD-like behavioral abnormalities and altered neuroinflammatory profiles, with an increase in pro-inflammatory cytokines and microglial markers. In addition to neuroinflammatory response, an abnormal expression of endogenous retroviruses (ERVs) has been identified in neurodevelopmental disorders and have been found to correlate with disease severity. Our aim was to evaluate the transcriptional profile of several ERV families, ERV-related genes, and inflammatory mediators (by RT real-time PCR) in mouse offspring of both sexes, prenatally exposed to polyinosinic:polycytidylic acid (Poly I:C), a synthetic double-stranded RNA molecule targeting TLR-3 that mimics viral maternal infection during pregnancy. We found that prenatal exposure to Poly I:C deregulated the expression of some ERVs and ERV-related genes both in the prefrontal cortex (PFC) and hippocampus, while no changes were detected in the blood. Interestingly, sex-related differences in the expression levels of some ERVs, ERV-related genes, and inflammatory mediators that were higher in females than in males emerged only in PFC. Our findings support the tissue specificity of ERV and ERV-related transcriptional profiles in MIA mice.

In our genomes there are thousands of copies of human endogenous retroviruses (HERVs) originated from the integration of exogenous retroviruses that infected germ line cells millions of years ago, and currently an altered expression of this elements has been associated to the onset, progression and acquisition of aggressiveness features of many cancers. The transcriptional reactivation of HERVs is mainly an effect of their responsiveness to some factors in cell microenvironment, such as nutrients, hormones and cytokines. We have already demonstrated that, under pressure of microenvironmental changes, HERV-K (HML-2) activation is required to maintain human melanoma cell plasticity and CD133+ cancer stem cells survival. In the present study, the transcriptional activity of HERV-K (HML-2), HERV-H, CD133 and the embryonic transcription factors OCT4, NANOG and SOX2 was evaluated during the in vitro treatment with antiretroviral drugs in cells from melanoma, liver and lung cancers exposed to microenvironmental changes. The exposure to stem cell medium induced a phenotype switching with the generation of sphere-like aggregates, characterized by the concomitant increase of HERV-K (HML-2) and HERV-H, CD133 and embryonic genes transcriptional activity. Although with heterogenic response among the different cell lines, the in vitro treatment with antiretroviral drugs affected HERVs transcriptional activity in parallel with the reduction of CD133 and embryonic genes expression, clonogenic activity and cell growth, accompanied by the induction of apoptosis. The responsiveness to antiretroviral drugs treatment of cancer cells with stemness features and expressing HERVs suggests the use of these drugs as innovative approach to treat aggressive tumours in combination with chemotherapeutic/radiotherapy regimens. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12307-019-00231-3) contains supplementary material, which is available to authorized users.

Background Despite an impressive effort in clinical research, no standard therapeutic approach for coronavirus disease 2019 (COVID-19) patients has been established, highlighting the need to identify early biomarkers for predicting disease progression and new therapeutic interventions for patient management. The present study aimed to evaluate the involvement of the human endogenous retrovirus -W envelope (HERV-W ENV) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection considering recent findings that HERVs are activated in response to infectious agents and lead to various immunopathological effects. We analysed HERV-W ENV expression in blood cells of COVID-19 patients in correlation with clinical characteristics and have discussed its potential role in the outcome of the disease. Methods We analysed HERV-W ENV expression in blood samples of COVID-19 patients and healthy donors by flow cytometry and quantitative reverse transcriptase PCR analysis, and evaluated its correlation with clinical signs, inflammatory markers, cytokine expression, and disease progression. Findings HERV-W ENV was highly expressed in the leukocytes of COVID-19 patients but not in those of healthy donors. Its expression correlated with the markers of T-cell differentiation and exhaustion and blood cytokine levels. The percentage of HERV-W ENV-positive lymphocytes correlated with inflammatory markers and pneumonia severity in COVID-19 patients. Notably, HERV-W ENV expression reflects the respiratory outcome of patients during hospitalization. Interpretation Given the known immuno- and neuro-pathogenicity of HERV-W ENV protein, it could promote certain pathogenic features of COVID-19 and therefore serve as a biomarker to predict clinical progression of disease and open to further studies for therapeutic intervention. Funding Information available at the end of the manuscript.

Background Coronavirus disease 2019 (COVID-19) is characterized by immune-mediated lung injury and complex alterations of the immune system, such as lymphopenia and cytokine storm, that have been associated with adverse outcomes underlining a fundamental role of host response in severe acute respiratory syndrome coronavirus 2 infection and the pathogenesis of the disease. Thymosin alpha 1 (Tα1) is one of the molecules used in the management of COVID-19, because it is known to restore the homeostasis of the immune system during infections and cancer. Methods In this study, we captured the interconnected biological processes regulated by Tα1 in CD8+ T cells under inflammatory conditions. Results Genes associated with cytokine signaling and production were upregulated in blood cells from patients with COVID-19, and the ex vivo treatment with Tα1-mitigated cytokine expression, and inhibited lymphocyte activation in a CD8+ T-cell subset specifically. Conclusion These data suggest the potential role of Tα1 in modulating the immune response homeostasis and the cytokine storm in vivo., Graphical Abstract Graphical Abstract, Tα1 restores dysregulated pathways under proinflammatory conditions. Tα1 inhibits cytokine-related gene expression in blood cells from COVID-19 individuals. Tα1 disrupts cytokines and T-cell activation in COVID-19. Modulation of CD38 in CD8+ T cells by Tα1 depends on the clinical score.

Background: Despite an impressive effort in clinical research, no standard therapeutic approach for coronavirus disease 2019 (COVID-19) patients has been established, highlighting the need to identify early biomarkers for predicting disease progression and new therapeutic intervention for patient management. The present study aimed to evaluate the involvement of the human endogenous retrovirus -W envelope (HERV-W ENV) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection considering recent findings that HERVs are activated in response to infectious agents and lead to various immunopathological effects. We analysed HERV-W ENV expression in blood cells of COVID-19 patients in correlation with clinical characteristics and have discussed its potential role in the outcome of the disease. Methods: We analysed HERV-W ENV expression in blood samples of Covid-19 patients and healthy donors by flow cytometry and quantitative reverse transcriptase PCR analysis, and evaluated its correlation with clinical signs, inflammatory markers, cytokine expression, and disease progression. Findings: HERV-W ENV was highly expressed in the leukocytes of COVID-19 patients but not in those of healthy donors. Its expression correlated with the markers of T-cell differentiation and exhaustion and blood cytokine levels. The percentage of HERV-W ENV-positive lymphocytes correlated with inflammatory markers and pneumonia severity in COVID-19 patients. Notably, HERV-W ENV expression reflects the respiratory outcome of patients during hospitalization. Interpretation: Given the known immuno- and neuro-pathogenicity of HERV-W ENV protein, it could promote certain pathogenic features of COVID-19 and therefore serve as a biomarker to predict clinical progression of disease and as a potential therapeutic target. Funding: Genero Innovation Declaration of Interests: C.M. reports grant from Gilead, outside the submitted work; L.S. reports personal fees and other from Merck, Gilead, and Abbvie, grants from Gilead, outside the submitted work; HP and BC receive compensation for their work by Geneuro-Innovation. The other authors declare no competing interests. Ethics Approval Statement: Ethical approval for the collection and use of human samples was obtained from the ethical board of the Hospital, COronaVIrus Disease: Safety and Efficacy of Experimental Treatment (COVID_SEET prot.7562/2020, 9th April 2020, experimental register 46.20).

Prenatal treatment with the antiepileptic drug valproic acid (VPA) is associated with a significant risk of somatic anomalies, neurodevelopmental delays, and 7-10× increase in the incidence of autism spectrum disorders (ASD) in children. Rodents exposed to VPA in pregnancy show birth defects, deficits in neurodevelopment, and cognitive/social anomalies resembling those of ASD children. Mechanisms of VPA neurobehavioral toxicity are still unclear but as VPA is a non-selective inhibitor of histone deacetylases, epigenetic modifications are likely involved. This study was aimed to evaluate the transgenerational impact of prenatal VPA exposure on mouse early behavioral development, studying F1, F2, and F3 generations after VPA challenge on gestational day (GD) 10.5. We also analyzed in brain and in peripheral blood mononuclear cells the expression levels of different endogenous retrovirus (ERV) families, potential biomarkers of derailed brain development, since human ERVs have been implicated in the pathogenesis of neurodevelopmental disorders (NDDs) such as ASD. Somatic effects of VPA were evident only in F1 generation and more markedly in the female sex. Across F1 and F2 generations, VPA delayed righting reflex, increased motor activity, and reduced ultrasonic vocalizations. The behavioral changes in F3 are milder though in the same direction. VPA increased expression of most ERVs across the three generations in brain and blood. In utero VPA induced neurodevelopmental alterations more marked in the maternal lineage that persisted also in F3, suggesting ERVs as possible downstream effectors of the VPA epigenetic alterations.

Thymosins have been extracted, characterized, and identified from Thymus. The Thymosins are hormones whose therapeuric applications have seen a recent increase. The action of Thymosin α1 is based on the stimulation of the immune response with a large number of results in a variety of pathologies. The absence of a specific receptor prompted us to investigate the direct interaction with membranes, particularly those exposing phosphatidylserine thus contributing to assess the Thymosin α1's pleiotropy.The interaction with membranes has been studied with a number of models indicating that Thymosin α1 interacts preferentially with negative regions of the membrane (SDS mixed with dodecylphosphocholine) or, better, with vesicles of dipalmitoylphosphatidylcholine with exposed phosphatidylserine.The study of the role of the membrane in the mechanism of action of Thymosin α1 indicated that probably the first interaction occurs at the membrane level with recognition of negative surface due to the phosphatidylserine exposure. Upon assuming a conformation, with two helices with a disordered tract in between, it diffuses on the membrane surface by lateral diffusion. Then the interaction with membrane receptor(s) causes a membrane complex to be formed, with an activation of a signalling cascade. This can be considered the basis of its pleiotropy. Differences in structuration mechamism of Thymosin β4 was outlined.

Emerging data indicate that the reverse transcriptase (RT) protein encoded by LINE-1 transposable elements is a promising cancer target. Nonnucleoside RT inhibitors, e.g. efavirenz (EFV) and SPV122.2, reduce proliferation and promote differentiation of cancer cells, concomitant with a global reprogramming of the transcription profile. Both inhibitors have therapeutic anticancer efficacy in animal models. Here we have sought to clarify the mechanisms of RT inhibitors in cancer cells. We report that exposure of PC3 metastatic prostate carcinoma cells to both RT inhibitors results in decreased proliferation, and concomitantly induces genome damage. This is associated with rearrangements of the nuclear architecture, particularly at peripheral chromatin, disruption of the nuclear lamina, and budding of micronuclei. These changes are reversible upon discontinuation of the RT-inhibitory treatment, with reconsititution of the lamina and resumption of the cancer cell original features. The use of pharmacological autophagy inhibitors proves that autophagy is largely responsible for the antiproliferative effect of RT inhibitors. These alterations are not induced in non-cancer cell lines exposed to RT inhibitors. These data provide novel insight in the molecular pathways targeted by RT inhibitors in cancer cells.

Human endogenous retroviruses (HERVs) are genetic elements resulting from relics of ancestral infection of germline cells, now recognized as cofactors in the etiology of several complex diseases. Here we present a review of findings supporting the role of the abnormal HERVs activity in neurodevelopmental disorders. The derailment of brain development underlies numerous neuropsychiatric conditions, likely starting during prenatal life and carrying on during subsequent maturation of the brain. Autism spectrum disorders, attention deficit hyperactivity disorders, and schizophrenia are neurodevelopmental disorders that arise clinically during early childhood or adolescence, currently attributed to the interplay among genetic vulnerability, environmental risk factors, and maternal immune activation. The role of HERVs in human embryogenesis, their intrinsic responsiveness to external stimuli, and the interaction with the immune system support the involvement of HERVs in the derailed neurodevelopmental process. Although definitive proofs that HERVs are involved in neurobehavioral alterations are still lacking, both preclinical models and human studies indicate that the abnormal expression of ERVs could represent a neurodevelopmental disorders-associated biological trait in affected individuals and their parents.

Thymosin α1 is a peptidic hormone with pleiotropic activity and is used in the therapy of several diseases. It is unstructured in water solution and interacts with negative regions of vesicles by assuming two tracts of helical conformation with a structural break between them. This study reports on Thymosin α1's interaction with mixed phospholipids phosphatidylcholine and phosphatidylserine, the negative component of the membranes, by ¹H and natural abundance ¹⁵N nuclear magnetic resonance (NMR). The results indicate that interaction occurs when the membrane is negatively charged by exposing phosphatidylserine. Moreover, the direct interaction of Thymosin α1 with K562 cells with an overexposure of phosphatidylserine as a consequence of resveratrol-induced apoptosis was conducted. Thymosin α1's interaction with human serum albumin was also investigated by NMR spectroscopy. Steady-state saturation transfer, transfer nuclear Overhauser effect spectroscopy, and diffusion-ordered spectroscopy methodologies all reveal that the C-terminal region of Thymosin α1 is involved in the interaction with serum albumin. These results may shed more light on Thymosin α1's mechanism of action by its insertion in negative regions of membranes due to the exposure of phosphatidylserine. Once Thymosin α1's N-terminus has been inserted into the membrane, the rest may interact with nearby proteins and/or receptors acting as effectors and causing a biological signaling cascade, thus exerting thymosin α1's pleiotropy.

Cancer incidence and mortality, metastasis, drug resistance and recurrence are still the critical issues of oncological diseases. In this scenario, increasing scientific evidences demonstrate that the activation of human endogenous retroviruses (HERVs) is involved in the aggressiveness of tumors such as melanoma, breast, germ cell, renal, ovarian, liver and haematological cancers. In their dynamic regulation, HERVs have also proved to be important determinants of pluripotency in human embryonic stem cells (ESC) and of the reprogramming process of induced pluripotent stem cells (iPSCs). In many types of tumors, essential characteristics of aggressiveness have been associated with the achievement of stemness features, often accompanied with the identification of defined subpopulations, termed cancer stem cells (CSCs), which possess stem cell-like properties and sustain tumorigenesis. Indeed, CSCs show high self-renewal capacity with a peculiar potential in tumor initiation, progression, metastasis, heterogeneity, recurrence, radiotherapy and drug resistance. However, HERVs role in CSCs biology is still not fully elucidated. In this regard, CD133 is a widely recognized marker of CSCs, and our group demonstrated, for the first time, the requirement of HERV-K activation to expand and maintain a CD133+ melanoma cell subpopulation with stemness features in response to microenvironmental modifications. The review will discuss HERVs expression as cancer hallmark, with particular focus on their role in the regulation of cancer stemness features and the potential involvement as targets for therapy.

Background: Thymosin alpha 1 (T?1) is a well-recognized immune response modulator in a wide range of disorders, particularly infections and cancer. The bioinformatic analysis of public databases allows drug repositioning, predicting a new potential area of clinical intervention. We aimed to decipher the cellular network induced by T?1 treatment to confirm present use and identify new potential clinical applications. Research design and methods: We used the transcriptional profile of human peripheral blood mononuclear cells treated in vitro with T?1 to perform the enrichment network analysis by the Metascape online tools and the disease enrichment analysis by the DAVID online tool. Results: Networked cellular responses reflected T?1 regulated biological processes including immune and metabolic responses, response to compounds and oxidative stress, ion homeostasis, peroxisome biogenesis and drug metabolic process. Beyond cancer and infections, the analysis evidenced the association with disorders such as kidney chronic failure, diabetes, cardiovascular, chronic respiratory, neuropsychiatric, neurodegenerative and autoimmune diseases. Conclusions: In addition to the known ability to promote immune response pathways, the network enrichment analysis demonstrated that T?1 regulates cellular metabolic processes and oxidative stress response. Notable, the analysis highlighted the association with several diseases, suggesting new translational implication of T?1 treatment in pathological conditions unexpected until now.

LINE-1 elements account for about 17% of the human genome and harbour two open reading frames: ORF1, encoding a 40 kDa RNA-binding protein, and ORF2, coding for a 150 kDa protein with reverse transcriptase (RT) activity. LINE-1s are highly expressed in embryos and tumor cells while being virtually silent in differentiated tissues and, consistently, both ORF-1p and ORF-2p have been detected in human cancers. RT-encoding ORF2 is expressed early in pre-neoplastic lesions suggesting that RT expression may be a potential cause, rather than a consequence, of cancer onset. Experimental data emerging from in vitro and in vivo studies confirm this view. Preclinical work showed that RT inhibition reduces proliferation, promotes differentiation of cancer cells and antagonizes tumor progression in murine models. Moreover, a recent phase II trial on metastatic hormone-resistant prostate cancer patients has confirmed the anticancer efficacy of RT inhibitors. Together, these data indicate that LINE-1-encoded RT emerges as a potential therapeutic target for a large spectrum of cancers and RT inhibitors as effective tools in a novel anti-cancer, non-cytotoxic, differentiation therapy.

LINE-1 (L1) retrotransposons are a source of endogenous reverse transcriptase (RT) activity, which is expressed as part of the L1-encoded ORF2 protein (L1-ORF2p). L1 elements are highly expressed in many cancer types but not in differentiated somatic tissues. We previously found that RT inhibition reduces cell proliferation and promote differentiation in neoplastic cells, indicating that high endogenous RT activity promote cancer growth. Here we investigate the expression of L1-ORF2p in several human types ocancer.We have developed a highly specific monoclonal antibody (mAb chA1-L1) raised against the endonuclease domain of L1-ORF2p as a tool to study ORF2p expression and localization in human cancer cell lines and staged bioptic tissues. We uncover new evidence for high levels of L1-ORF2p in transformed cell lines and staged epithelial cancer tissues (colon, prostate, lung and breast) while no or only basal ORF2p expression was detected in non-transformed cells. An in-depth analysis of colon and prostate cancer tissues shows ORF2p expression in pre-neoplastic stages, namely transitional mucosa and prostate intraepithelial neoplasia (PIN), respectively. Our results show that L1-ORF2p is overexpressed in tumor tissues and in pre-neoplastic colon and prostate tissues; this latter finding suggests that ORF2p could be considered as a potential early cancer diagnostic biomarker.

Background and aims: The immunomodulatory activity of thymosin a1 (Ta1) on innate immunity has been extensively described, but its mechanism of action is not completely understood. We explored the possibility that Ta1-stimulation could affect the formation of podosomes, the highly dynamic, actin-rich, adhesion structures involved in macrophage adhesion/ 15 chemotaxis. Methods: The following methods were used: optical and scanning electron microscopy for analyzing morphology of human monocyte-derived macrophages (MDMs); time-lapse imaging for visualizing the time-dependent modifications induced at early times by Ta1 treatment; confocal microscopy 20 andWestern blot for analyzing localization and expression of podosome components; and Matrigel Migration Assay and zymography for testing MDM invasive ability and metalloproteinase secretion. Results: We obtained data to support that Ta1 could affect MDM motility, invasion and chemotaxis by promptly stimulating assembly and disassembly 25 of podosomal structures. At very early times after its addition to cell culture medium and within 1 h of treatment, Ta1 induces modifications in MDM morphology and in podosomal components that are suggestive of increased podosome turnover. Conclusions: Since impairment of podosome formation leads to reduced 30 innate immunity and is associated with several immunodeficiency disorders, we confirm the validity of Ta1 as a potent activator of innate immunity and suggest possible new clinical application of this thymic peptide.

Thymosin α-1 (Tα1) exploits a specific action on lymphoid cells and is able to induce in peripheral blood mononuclear cells (PBMCs) a strong transcriptional response. CD8 antiviral factor activity plays a role in the control or prevention of HIV-1 infection by a non-cytolytic mechanism. The ability of Tα1 to modulate the release of antiretroviral soluble factors by CD8(+) cells was investigated.Supernatants from lipopolysaccharide (LPS) stimulated CD8(+)-isolated cells treated with Tα1 were screened on in vitro infection of human monocyte-derived macrophages (MDMs) and PBMCs with HIV-1, and of PBMCs with human T lymphotropic virus 1 (HTLV-1). In CD8(+) cells, as well as in PBMCs of healthy donors as from HIV(+) individuals, a microarray analysis to assess the transcriptional response after treatment was performed.Tα1 potentiates the release, in LPS-stimulated CD8(+) cells, of soluble factors able to inhibit both in vitro HIV-1 infection of MDMs and PBMCs and in vitro HTLV-1 infection of PBMCs. A distinctive transcriptional profile was induced by Tα1 in PBMCs from HIV(+) donors.These findings suggest that Tα1 would represent a re-evaluated approach to antiretroviral therapy in combination with innovative treatments and with vaccine administration.

Retroelements, such as Human Endogenous Retroviruses (HERVs), have been implicated in many complex diseases, including neurological and neuropsychiatric disorders. Previously, we demonstrated a distinctive expression profile of specific HERV families in peripheral blood mononuclear cells from Autistic Spectrum Disorders (ASD) patients, suggesting their involvement in ASD. Here we used two distinct ASD mouse models: inbred BTBR T+tf/J mice and CD-1 outbred mice prenatally exposed to valproic acid. Whole embryos, blood and brain samples from the offspring were collected at different ages and the expression of several ERV families (ETnI, ETnII-α, ETnII-β, ETnII-γ, MusD and IAP), proinflammatory cytokines (IL-1β, IL-6 and TNF-α) and Toll-like receptors (TLR3 and TLR4) was assessed. In the two distinct mouse models analysed, the transcriptional activity of the ERV families was significant higher in comparison with corresponding controls, in whole embryos, blood and brain samples. Also the expression levels of the proinflammatory cytokines and TLRs were significantly higher than controls. Current results are in agreement with our previous findings in ASD children, supporting the hypothesis that ERVs may serve as biomarkers of atypical brain development. Moreover, the changes in ERVs and proinflammatory cytokines expression could be related with the autistic-like traits acquisition in the two mouse models.

Thymosin α1 (Tα1) is a peptide hormone whose therapeutic application has been approved in several diseases, but the description of a precise receptor for its therapeutic action still remains elusive and some knowledge of the mechanism of interaction with the cell membrane still needs to be clarified. This work is aimed at studying the folding and interaction of Tα1, which is completely unstructured in water solution, with model membranes.The folding and interaction of Tα1 with sodium dodecyl sulfate micelles was monitored by NMR and CD spectroscopy techniques.Tα1 assumes a helical conformation in the presence of sodium dodecyl sulfate micelles, showing a helical fold with a structural break around residues 9 and 14. These results were confirmed by circular dichroism and NMR spectroscopy. Moreover, by paramagnetic NMR relaxation it was found that Tα1 is inserted in the hydrophobic region of the micelles by the residues 1 - 5 of the N-terminal end. This result clarifies the modality of insertion that was not obtained in previous NMR studies in trifluoroethanol.These findings suggest that Tα1 folds on the membrane and, when inserted, may be able to interact with nearby proteins and/or receptors acting as an effector and causing a biological signaling cascade.

Human endogenous retroviruses (HERVs) have been implicated in human physiology and in human pathology. A better knowledge of the retroviral transcriptional activity in the general population and during the life span would greatly help the debate on its pathologic potential. The transcriptional activity of four HERV families (H, K, W, and E) was assessed, by qualitative and quantitative PCR, in PBMCs from 261 individuals aged from 1 to 80 years. Our results show that HERV-H, HERV-K, and HERV-W, but not HERV-E, are transcriptionally active in the test population already in the early childhood. In addition, the transcriptional levels of HERV-H, HERV-K, and HERV-W change significantly during the life span, albeit with distinct patterns. Our results, reinforce the hypothesis of a physiological correlation between HERVs activity and the different stages of life in humans. Studies aiming at identifying the factors, which are responsible for these changes during the individual’s life, are still needed. Although the observed phenomena are presumably subjected to great variability, the basal transcriptional activity of each individual, also depending on the different ages of life, must be carefully considered in all the studies involving HERVs as causative agents of disease.

Thymosin α1 (Tα1), is a peptidic hormone, whose immune regulatory properties have been demonstrated both in vitro and in vivo and approved in different countries for treatment of several viral infections and cancers. Tα1 assumes a conformation in negative membranes upon insertion into the phosphatidylserine exposure as found in several pathologies and in apoptosis. These findings are in agreement with the pleiotropy of Tα1, which targets both normal and tumor cells, interacting with multiple cellular components, and have generated renewed interest in the topic. Hyaluronan (HA) occurs ubiquitously in the extracellular matrix and on cell surfaces and has been related to a variety of diseases, and developmental and physiological processes. Proteins binding HA, among them CD44 and the Receptor for HA-mediated motility (RHAMM) receptors, mediate its biological effects. NMR spectroscopy indicated preliminarily that an interaction of Tα1 with HA occurs specifically around lysine residues of the sequence LKEKK of Tα1 and is suggestive of a possible interference of Tα1 in the binding of HA with CD44 and RHAMM. Further studies are needed to deepen these observations because Tα1 is known to potentiate the T-cell immunity and anti-tumor effect. The binding inhibitory activity of Tα1 on HA-CD44 or HA-RHAMM interactions can suppress both T-cell reactivity and tumor progression.

Thymosin α1 (Tα1) is a naturally occurring thymic peptide used worldwide in clinical trials for the treatment of infectious diseases and cancer. The immunomodulatory activity of Tα1 on innate immunity effector cells has been extensively described, but its mechanism of action is not completely understood. We report that Tα1-exposed human monocyte-derived macrophages (MDMs) assume the typical activated morphology also exhibited by lipopolysaccharide-activated MDMs, but show a comparatively higher ability of internalizing fluorescent beads and zymosan particles. Tα1 exposure also promptly and dramatically stimulates MDM phagocytosis and killing of Aspergillus niger conidia starting as soon as 30 min after challenge. The effect is dose dependent and early coupled to low transcription of the proinflammatory cytokines tumor necrosis factor α and interleukin-6 and unmodified Toll-like receptor expression. The Tα1-stimulated phagocytosis is strictly dependent on the integrity of the microtubule network and protein kinase C activity and occurs by a variation in the classic zipper model, with recruitment of vinculin and actin at the phagosome exhibiting a punctate distribution. These findings indicate that, in human mature MDMs, Tα1 implements pathogen internalization and killing via the stimulation of the complement receptor-mediated phagocytosis. Our observations document that Tα1 is an early and potent activator of innate immunity and reinforce the concept of its pleiotropy.

In spite of the consistent benefits for HIV-1 infected patients undergoing antiretroviral therapy, a complete immune reconstitution is usually not achieved. Actually, antiretroviral therapy may be frequently accompanied by immunological unresponsiveness, persistent inflammatory conditions and inefficient cytotoxic T-cell response. Thymosin alpha 1 is a thymic peptide that demonstrates a peculiar ability to restore immune system homeostasis in different physiological and pathological conditions (i.e., infections, cancer, immunodeficiency, vaccination and aging) acting as multitasking protein depending on the host state of inflammation or immune dysfunction. This review reports the present knowledge on the in vitro and in vivo studies concerning the use of thymosin alpha 1 in HIV-1 infection. Recent findings and future perspectives of therapeutic intervention are discussed.

Foodborne disease caused by Salmonella represents a worldwide public health problem. In Europe, salmonellosis is still the second most commonly recorded zoonosis. Since the standard culture method for detecting Salmonella (ISO 6579:2002) requires up to 5 days to produce results, the need to develop rapid methods represents an important issue for the authorities and the producers. The aim of the present study was the in-house validation, according to ISO 16140, of an open-formula diagnostic real-time PCR for the detection of Salmonella in all the different meat categories reported in the EU Regulations relative to microbiological criteria for food safety. The assay employed specific primers and a probe target within the ttrRSBCA locus, which allows the tetrathionate respiration in Salmonella. Selectivity, relative accuracy, relative sensitivity and relative specificity were established by testing 110 bacterial strains and 175 various edible meat samples. Results showed 100 % selectivity, 100 % relative accuracy, 100 % relative sensitivity and 100 % relative specificity of the real-time PCR when compared to the standard culture method used as reference. In addition, in order to minimize the effect of the competitive micro-flora naturally present on meat samples, a highly nutritious and selective commercial medium (ONE Broth Salmonella, Oxoid) was evaluated in comparison with the classical non-selective pre-enrichment broth (buffered peptone water). Results demonstrated that the ONE Broth Salmonella medium increases the growth of Salmonella in the presence of competitive micro-flora.

The innate immune response and its cellular effectors-peripheral blood mononuclear cells and differentiated macrophages-play a crucial role in detection and elimination of pathogenic microorganisms. Chemotherapy and some immunosuppressive drugs used after organ transplantation and for treatment of autoimmune diseases have, as main side effect, bone marrow suppression, which can lead to a reduced response of the innate immune system. Hence, many immune-depressed patients have a higher risk of developing bacterial and invasive fungal infections compared with immune-competent individuals. Thymosin α1 (Tα1) immunomodulatory activity on effector cells of the innate immunity has been extensively described, even if its mechanism of action is not completely understood. Here, we report some of the main knowledge on this topic, focusing on our in vitro and in vivo work in progress that reinforce the validity of Tα1 as a stimulatory agent for detection and elimination of pathogens by differentiated macrophages and for restoring immune parameters after chemotherapy-induced myelosuppression.

Since it was first identified, thymosin alpha 1 (Tα1) has been characterized to have pleiotropic effects on several pathological conditions, in particular as a modulator of immune response and inflammation. Several properties exerted by Tα1 may be attributable to a direct action on lymphoid cells. Tα1 has been shown to exert an immune modulatory activity on both T cell and natural killer cell maturation and to have an effect on functions of mature lymphocytes, including stimulating cytokine production and cytotoxic T lymphocyte-mediated cytotoxic responses. In previous studies we have shown that Tα1 increases the expression of major histocompatibility complex class I surface molecules in murine and human tumor cell lines and in primary cultures of human macrophages. In the present paper, we describe preliminary data indicating that Tα1 is also capable of increasing the expression of tumor antigens in both experimental and human tumor cell lines. This effect, which is exerted at the level of the target tumor cells, represents an additional factor increasing the antitumor activity of Tα1.

Two large families of retrotransposons, that is, LINE-1 (Long Interspersed Nuclear Elements-1) and endogenous retroviruses, encode reverse transcriptase (RT) proteins in vertebrates. We previously showed that mouse preimplantation embryos are endowed with an endogenous, functional RT activity. Inhibiting that activity by microinjecting antisense oligonucleotides against a highly active LINE-1 family member in mouse oocytes blocked developmental progression between the two- and four-blastomere stages, indicating that LINE-1-encoded RT activity is strictly required at this critical transition in early development. Here we show that incubation of mouse zygotes with 5'-bromodeoxyuridine (BrdU) yields massive incorporation of this nucleoside analogue in newly synthesized DNA; surprisingly, a significant incorporation still occurs in both zygotic pronuclei in the presence of aphidicolin, a specific inhibitor of DNA replication. This aphidicolin-resistant BrdU incorporation is quantitatively abolished when embryos are simultaneously exposed to abacavir, a nucleoside RT inhibitor, indicating its retrotranscription-dependent nature. Moreover, quantitative PCR analysis revealed a burst of new synthesis of LINE-1 copies at the zygote- and two-cell embryo stages. These findings support the conclusion that RT-dependent amplification of LINE-1 retrotransposons is a distinctive feature of early embryonic genomes. Its physiological involvement in preimplantation murine development is discussed.

LINE-1 (Long Interspersed Nuclear Elements) and HERVs (Human Endogenous Retroviruses) are two families of autonomously replicating retrotransposons that together account for about 28% of the human genome. Genes harbored within LINE-1 and HERV retrotransposons, particularly those encoding the reverse transcriptase (RT) enzyme, are generally expressed at low levels in differentiated cells, but their expression is upregulated in transformed cells and embryonic tissues. Here we discuss a recently discovered RT-dependent mechanism that operates in tumorigenesis and reversibly modulates phenotypic and functional variations associated with tumor progression. Downregulation of active LINE-1 elements drastically reduces the tumorigenic potential of cancer cells, paralleled by reduced proliferation and increased differentiation. Pharmacological RT inhibitors (e.g., nevirapine and efavirenz) exert similar effects on tumorigenic cell lines, both in culture and in animal models. The HERV-K family play a distinct complementary role in stress-dependent transition of melanoma cells from an adherent, non-aggressive, to a non-adherent, highly malignant, growth phenotype. In synthesis, the retrotransposon-encoded RT is increasingly emerging as a key regulator of tumor progression and a promising target in a novel anti-cancer therapy.

Thymosin alpha-1 (Talpha1) is a molecule retaining pleiotropic effects toward several pathological conditions, especially acting as a modulator of immune response and inflammation. However, molecular mechanisms underlying the peculiar, wide-range activity of Talpha1 have not been completely elucidated. To get information on the mechanism of action of Talpha1 and to find new potential applications, we performed the analysis of the transcription profile of 8300 genes in human peripheral blood mononuclear cells in vitro treated with Talpha1. Together with a variety of genes already known to be modulated by Talpha1, our analysis revealed a remarkable number of genes not yet described before to be modulated by this thymic peptide, revealing a complex scenario that underlines the role of this peptide as a regulator of lymphocyte functions. Here, we report the list of the more representative modulated genes and discuss their implications on the modulation of the innate and adaptive responses. Our results open new perspectives on the use of Talpha1 for the treatment of diseases associated with immune-disregulation.

Historically, natural products have represented a significant source of anticancer agents, with plant-derived drugs becoming increasingly explored. In particular, sanguinarine is a benzophenanthridine alkaloid obtained from the root of Sanguinaria canadensis, and from other poppy Fumaria species, with recognized anti-microbial, anti-oxidant and anti-inflammatory properties. Recently, increasing evidence that sanguinarine exibits anticancer potential through its capability of inducing apoptosis and/or antiproliferative effects on tumor cells, has been proved. Moreover, its antitumor seems to be due not only to its pro-apoptotic and inhibitory effects on tumor growth, but also to its antiangiogenic and anti-invasive properties. Although the precise mechanisms underlying the antitumor activity of this compound remain not fully understood, in this review we will focus on the most recent findings about the cellular and molecular pathways affected by sanguinarine, together with the rationale of its potential application in clinic. The complex of data currently available suggest the potential application of sanguinarine as an adjuvant in the therapy of cancer, but further pre-clinical studies are needed before such an antitumor strategy can be effectively translated in the clinical practice.

Thymosinα1 is a peptidic hormone with pleiotropic activity, which is used in the therapy of several diseases. It is unstructured in water solution and interacts with negative regions of micelles and vesicles assuming two tracts of helical conformation with a structural flexible break in between. The studies of the interaction of Thymosinα1 with micelles of mixed dipalmitoylphosphatidylcholine and sodium dodecylsulfate and vesicles with mixed dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylserine, the latter the negative component of the membranes, by (1)H and natural abundance (15)N NMR are herewith reported, reviewed, and discussed. The results indicate that the preferred interactions are those where the surface is negatively charged due to sodium dodecylsulfate or due to the presence of dipalmitoylphosphatidylserine exposed on the surface. In fact the unbalance of dipalmitoylphosphatidylserine on the cellular surface is an important phenomenon present in pathological conditions of cells. Moreover, the direct interaction of Thymosinα1 with K562 cells presenting an overexposure of phosphatidylserine as a consequence of resveratrol-induced apoptosis was carried out.

Thymosin a1 (Ta1) is a naturally occurring polypeptide that regulates immune cell development and function, and is also capable of interacting with multiple target cells with relevant biological effects. The rationale of Ta1 use in cancer treatment stems from the consideration that tumor progression is favored by a failure of the immune response and in turn induces immune suppression. This paper will review the historical background of Ta1 use in oncology, aiming to highlight the importance of Ta1 as an immunotherapeutic tool to be used in combination with chemotherapy, a concept that is not yet fully established in clinic. Areas covered: The efficacy and safety of combining Ta1 with chemotherapy and cytokines were first evaluated in murine tumor models, providing essential information about effects, mechanisms of action, doses and treatment protocols. The therapeutic potential of the chemo-immunotherapy protocol on metastatic melanoma and lung cancer has been confirmed in controlled clinical trials. Critical for the efficacy of the chemo-immunotherapy protocol is the dual action of Ta1 on immune effector and tumor cells. Expert opinion: On the basis of the preclinical and clinical results available, the use of the chemo-immunotherapy protocol, in which the role of Ta1 is central, is strongly recommended

Thymosin α1 is a peptidic hormone already used in the therapy of several diseases. Until now, the description of the precise receptor and mechanism for its action still remains elusive. The interaction of Thymosin α1, which is unstructured in water solution, has been recently studied in sodium dodecylsulphate micellar systems and it was reported that Thymosin α1 inserts in micelle assuming a conformation with two tracts of helix with a structural break in between. An investigation of its interaction both with micelles of dodecylphosphocholine alone and with mixed dodecylphosphocholine-sodium dodecylsulphate micelles is here reported. In these environments the results indicate that Thymosin α1 in phospholipidic membrane exposing choline polar heads interacts by aspecific modality and, oppositely, in the mixed dodecylphosphocholine-sodium dodecylsulphate micelles an insertion in the micellar hydrophobic region conformationally similar to that found in sodium dodecylsulphate micelles occurs. In presence of mixed micelles the insertion and structuration occur in preferred regions when the membrane models are negatively charged. From the point of view of the mechanism of action, insertion its N terminus in negative regions of membrane led to hypothesize that this process would be similar to a binding to phosphatidylserine exposed like in apoptotic cells. Thymosin α1 when inserted may interact with nearby proteins and/or receptors acting as effector and causing a biological signaling cascade. The recent attention to the phosphatidylserine exposure in cells may enforce the interest for these findings.

Undifferentiated cells and embryos express high levels of endogenous non-telomerase reverse transcriptase (RT) of retroposon/retroviral origin. We previously found that RT inhibitors modulate cell growth and differentiation in several cell lines. We have now sought to establish whether high levels of RT activity are directly linked to cell transformation. To address this possibility, we have employed two different approaches to inhibit RT activity in melanoma and prostate carcinoma cell lines: pharmacological inhibition by two characterized RT inhibitors, nevirapine and efavirenz, and downregulation of expression of RT-encoding LINE-1 elements by RNA interference (RNAi). Both treatments reduced proliferation, induced morphological differentiation and reprogrammed gene expression. These features are reversible upon discontinuation of the anti-RT treatment, suggesting that RT contributes to an epigenetic level of control. Most importantly, inhibition of RT activity in vivo antagonized tumor growth in animal experiments. Moreover, pretreatment with RT inhibitors attenuated the tumorigenic phenotype of prostate carcinoma cells inoculated in nude mice. Based on these data, the endogenous RT can be regarded as an epigenetic regulator of cell differentiation and proliferation and may represent a novel target in cancer therapy.

Melanoma is a widespread cancer with poor prognosis. Female hormones are known to be capable of influencing melanoma progression but clinical data related to pregnancy, oral contraception and hormone replacement therapy are controversial. A few reports show that in vitro progesterone (PG) affects melanoma growth in nuclear progesterone receptor (nPR)-positive and nPR-negative cells, but the experimental protocols used are quite different and the results are not univocal. Further research on this topic is thus needed especially in view of the widespread use of PG in clinical practice. In this study, we used human melanoma cells (A-375), which were cultured in vitro in the presence or absence of a wide range of PG concentrations (from 0.01 to 1000 M) in single treatment. Daily cell count, cell cycle analysis and apoptosis assay were performed. Our results show that the low PG concentrations (from 0.01 to 1.0 M) promote a significant increase of melanoma cell proliferation but this growth-stimulatory effect is not observed at 10 M PG and the higher PG concentrations (i.e. 100 and 1000 M) induce a cell density reduction which is the result of both cell cycle arrest and apoptosis. These findings confirm and extend previous observations reported in the international literature. A higher caution in the clinical use of progesterone is thus mandatory, since PG concentrations capable of stimulating melanoma cell proliferation are very close to those commonly used in a wide spectrum of physio-pathological conditions.

One of the proposed mechanisms for multidrug resistance relies on the ability of resistant tumor cells to efficiently promote glutathione S -transferase (GST)-catalyzed GSH conjugation of the antitumor drug. This type of conjugation, observed in several families of drugs, has never been documented satisfactorily for anthracyclines. Adriamycin-resistant human breast cancer MCF-7/DOX cells, presenting a comparable GSH concentration, but a 14-fold increase of the GST P1–1 activity relative to the sensitive MCF-7 cells, have been treated with adriamycin in the presence of verapamil, an inhibitor of the 170 P-glycoprotein (P-gp) drug transport protein, and scrutinized for any production of GSH–adriamycin conjugates. HPLC analysis of cell content and culture broths have shown unequivocally that no GSH conjugates are present either inside the cell or in the culture broth. The only anthracycline present inside the cells after 24 hr of incubation was > 98% pure adriamycin. Confocal laser scanning microscopic observation showed that in MCF-7/DOX cells adriamycin was localized mostly in the Golgi apparatus rather than in the nucleus, the preferred site of accumulation for sensitive MCF-7 cells. These findings rule out GSH conjugation or any other significant biochemical transformation as the basis for resistance to adriamycin and as a ground for the anomalous localization of the drug in the cell. Adriamycin, daunomycin, and menogaril did not undergo meaningful conjugation to GSH in the presence of GST P1–1 at pH 7.2. Indeed, their synthetic C(7)-aglycon–GSH conjugates exerted a strong inhibitory effect on GST P1–1, with K i at 25° in the 1–2 μM range, scarcely dependent on their stereochemistry at C(7).

This study examines the viability and functionality of two insulinoma cell lines, RIN (1048) and betaTC6F7, encapsulated within microfabricated biocapsules. Surface and bulk micromachining are integrated in the biocapsule fabrication process, resulting in a diffusion membrane with uniform pore size distribution as well as mechanical and chemical stability, surrounded by an anisotropically-etched silicon wafer, which serves as the encapsulation cavity. Insulinoma cells (4500 cells/biocapsule) were enclosed within these microfabricated biocapsules and subjected to a static incubation study after either implantation in BALB-C mice or incubation in vitro. Examination of retrieved microfabricated biocapsules revealed an insulin stimulatory index of approximately 1.5 for encapsulated RIN cells and 3.6 for encapsulated betaTC6F7 cells for biocapsules with 18 nm pore sized microfabricated membranes, similar to indices of biocapsules incubated in vitro. There was an 80% decrease in cell stimulatory response between in vitro and in vivo 66 nm-biocapsules as compared to 20% for 18 nm-biocapsules, indicating that the immunoisolatory effectiveness depends greatly on achieving uniform pore sizes in the size range of 18 nm or smaller. The present study demonstrates the feasibility of using microfabricated biocapsules for the immunoisolation of insulinoma cells lines. The microfabricated biocapsule may serve as an alternative to conventional polymeric based biocapsules for possible use as in vivo insulin secreting bioreactor.

CaMBr1 is a blood group-related tumour-associated antigen, whose pattern of expression provides a therapeutic window for passive or active immunotherapy and points to the promise of a vaccine against carcinomas overexpressing this antigen. In this context, an animal model that closely mimics the human situation would be extremely useful. We, therefore, utilised the murine monoclonal antibody MBr1, which defines CaMBr1, as a useful probe to detect the molecule targeted for vaccine development on canine and feline spontaneous breast and uterus tumours and on their normal counterparts, and on rat normal tissues and carcinoma cell lines. Immunoperoxidase staining of cryostat sections revealed homogeneous CaMBr1 expression only in normal feline uterus and a uterus papilloma, whereas MBr1 reactivity was very weak and heterogeneous in normal (1/3 and 1/3) and tumour (1/10 and 1/6) breast tissues from dogs and cats, respectively. In contrast, the data obtained in rat tissues were reproducible in the strains tested and showed that CaMBr1 was expressed in all epithelial tissues of the digestive tract, although with variable intensities. Monoclonal antibody staining appeared to correspond to membrane-bound structures as well as mucinous secretions. Similarly, secretion products of lactating mammary glands expressed CaMBr1. The spectrum of expression on rat digestive tract was broader than that in humans but the specificity of MBr1 reactivity was confirmed by competition assay with a synthetic tetrasaccharide that mimics the CaMBr1 antigen. On FACS analysis, only one of two clonal derivatives of the rat breast carcinoma line RAMA 25 expressed CaMBr1, and a negative cell subset was evident in repeated experiments. By contrast, both colon carcinoma lines, DHD/K12 and CC531, showed staining with MBr1, albeit at different levels of intensity, and no evidence of a negative subset. The cell line CC531 maintained or even increased CaMBr1 expression levels following transplantation in syngeneic immunocompetent animals. Our data suggest the usefulness of the rat as a test model for vaccines against human cancers overexpressing the CaMBr1 antigen.